Ready-to-use bivalirudin compositions

ABSTRACT

Ready-to-use bivalirudin compositions, methods of using the ready-to-use bivalirudin compositions, and methods of preparing the ready-to-use bivalirudin compositions. The ready-to-use bivalirudin compositions comprise bivalirudin and one or more stabilizing agents. The one or more stabilizing agents may be buffering agents having a pKa of about 2.5 to about 6.5, pH-adjusting agents, polymers, preservatives, antioxidants, sugars or polyols, or a combination thereof. The ready-to-use bivalirudin compositions may also comprise [9-10]-cycloimido bivalirudin, [11-12]-cycloimido bivalirudin, or a combination thereof. The method of using the ready-to-use bivalirudin compositions comprises administering the ready-to-use compositions to a patient in need thereof. Further, the method of preparing the ready-to-use bivalirudin compositions comprises mixing bivalirudin with one or more stabilizing agents.

INCORPORATION BY REFERENCE

All documents cited or referenced herein (“herein cited documents”), andall documents cited or referenced in herein cited documents, togetherwith any manufacturer's instructions, descriptions, productspecifications, and product sheets for any products mentioned herein orin any document incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention.

FIELD OF THE INVENTION

The present invention is generally directed towards stable, ready-to-use(“RTU”) compositions comprising bivalirudin and one or more stabilizingagents, whereby the RTU composition has a pH of about 4 to less than 5.In certain embodiments, the one or more stabilizing agents may bebuffering agents, pH-adjusting agents, polymers, preservatives,antioxidants, sugars or polyols, or a combination thereof. In someembodiments, the RTU bivalirudin compositions may comprise[9-10]-cycloimido bivalirudin, [11-12]-cycloimido bivalirudin, or acombination thereof.

The present invention is also generally directed towards a method ofpreparing an RTU bivalirudin composition. In various embodiments of theinvention, this method may comprise mixing bivalirudin with one or morestabilizing agents to obtain an RTU composition that has a pH of about 4to less than 5. In another embodiment of the present invention, themethod may comprise mixing bivalirudin with one or more stabilizingagents and adjusting the pH to about 4 to less than 5.

Further, the present invention is generally directed towards a method oftreating a patient in need thereof with the RTU bivalirudin composition.In certain embodiments, this method comprises administering the RTUbivalirudin composition to a patient in need thereof.

BACKGROUND OF THE INVENTION

Anticoagulants are substances that prevent blood from clotting. They arecommonly used during percutaneous coronary intervention (“PCI”) andother catherization techniques in order to reduce bleeding complicationsduring surgery. One class of anticoagulants is direct thrombininhibitors that disrupt the activity of thrombin, a serine proteaseinvolved in the coagulation cascade that initiates clotting whenfibrinogen is converted to fibrin. Thrombin also activates Factor XIIIinto Factor XIIIa (the latter which links fibrin polymers covalently),Factors V and VIII (which promote thrombin generation), and platelets(which help propagate the thrombus).

Bivalirudin directly inhibits thrombin by specifically binding to bothits catalytic site and anion-binding exosite, and is regarded as ahighly effective anticoagulant for use during catherization procedures.Bivalirudin, also known as hirulog, is a synthetic congener of thenaturally occurring thrombin peptide inhibitor hirudin, which is foundin the saliva of the medicinal leech Hirudo medicinalis. Hirudinconsists of 65 amino acids, although shorter peptide segments haveproven to be effective as thrombin inhibitors. U.S. Pat. No. 5,196,404(“'404 patent”, incorporated herein by reference) discloses bivalirudinamong these shorter peptides that demonstrate anticoagulant activity.However, in contrast to hirudin, bivalirudin is a reversible thrombininhibitor that is ideal for temporary prevention of blood clottingduring catherization procedures.

Bivalirudin is a synthetic 20 amino acid peptide having the chemicalname ofD-Phenylalanyl-L-Prolyl-L-Arginyl-L-Prolyl-Glycyl-Glycyl-Glycyl-Glycyl-L-Asparagyl-Glycyl-L-Aspartyl-L-Phenylalanyl-L-Glutamyl-L-Glutamyl-L-Isoleucyl-L-Prolyl-L-Glutamyl-L-Glutamyl-L-Tyrosyl-L-Leucinetrifluoroacetate (salt) hydrate and has a molecular weight of 2180daltons (as the free base form). Bivalirudin is made up of the aminoacid sequence:(D-Phe)-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu(SEQ ID NO: 1). As used herein, the term bivalirudin refers to thepeptide comprising SEQ ID NO: 1, and salts thereof.

Bivalirudin can be formulated into a lyophilized drug product such asAngiomax®. Approved indications for Angiomax® include treatment inpatients with unstable angina undergoing percutaneous transluminalcoronary angioplasty (“PTCA”); administration with the provisional useof glycoprotein IIb/IIIa inhibitor for use as an anticoagulant inpatients undergoing PCI; and treatment in patients with, or at risk of,heparin-induced thrombocytopenia (“HIT”) or heparin-inducedthrombocytopenia and thrombosis syndrome (“HITTS”) undergoing PCI. SeeAngiomax® Prescribing Information.

At present, bivalirudin is solely available in the form of a lyophilizedcomposition that must be reconstituted prior to administration. Ingeneral, to administer a lyophilized drug composition, multiple stepsare required as the lyophilized cake is first reconstituted, diluted andthen administered. In some cases with certain drugs, the completedissolution of the powder may require prolonged shaking. Lack ofcomplete dissolution of certain drug powders can result in suboptimaldosing to the patient, which may result in decreased efficacy. Moreover,reconstitution introduces the potential for calculation and dilutionerrors. See generally Fanikos et al., Am. J. Cardio., 94 (2004) 532-535.

Other drugs that are marketed as RTU products are readily available forpatient administration and do not require lengthy preparation. As such,these products lead to more efficient use of hospital resources andfewer mixing mistakes and dosing errors. See generally Joint Commission,2009 National Patient Safety Goals Manual Chapter; Joint Commission,Sentinel Event Alert Issue 41, Sep. 24, 2008, Preventing Errors Relatingto Commonly Used Anticoagulants. These RTU compositions give thephysicians and clinicians assurance of the appropriate drugconcentration, allowing them to focus on the treatment without concernfor the logistics around drug preparation. Thus, patients withcardiovascular disease, who require an immediate invasive intervention,would benefit from an RTU bivalirudin composition.

SUMMARY OF THE INVENTION

The present invention relates to an RTU bivalirudin compositioncomprising bivalirudin and one or more pharmaceutically acceptablestabilizing agents. In some embodiments, the composition has a pH ofabout 4 to less than 5. In certain embodiments, the total impurities areless than about 15% area-under-the-curve (“AUC”) after storage at 25° C.for 1 month.

In embodiments of the present invention, the one or more stabilizingagents may comprise buffering agents having a pKa of about 2.5 to about6.5, such as acetate, tartrate, ascorbate, lactobionate, gentisate,succinate, lactate, α-lipoic acid, or any combination thereof;pH-adjusting agents such as acetic acid, sodium hydroxide, or anycombination thereof; polymers, such as polyethylene glycol, poloxamer,polysorbates, hydroxyethyl starch, polyvinylpyrrolidone, or anycombination thereof; preservatives, such as methyl-, ethyl- andpropyl-parabens or any combinations thereof; antioxidants such ashistidine, methionine, or any combination thereof; or sugars or polyols,such as sucrose, dextrose, dextrin, propylene glycol, sorbitol,glycerol, or any combination thereof.

In various embodiments, the composition may further comprise one or moretonicity agents such as inorganic salts, organic salts, or a combinationthereof. The inorganic salts may comprise sodium chloride, potassiumchloride, magnesium chloride, or calcium chloride, and the organic saltsmay comprise conjugate bases of trifluoroacetic acid.

In some embodiments of the present invention, the composition mayfurther comprise [9-10]-cycloimido bivalirudin and/or [11-12]-cycloimidobivalirudin. After storage at 25° C. for 1 month, the [9-10]-cycloimidobivalirudin may be greater than 0.2% AUC and less than about 5% AUC, andthe [11-12]-cycloimido bivalirudin may be greater than 0.1% AUC and lessthan about 5% AUC.

In certain embodiments of the present invention, the bivalirudin ispresent in an amount of about 1 mg/mL to about 10 mg/mL, or about 5mg/mL.

In embodiments of the present invention, the RTU bivalirudin compositioncomprises bivalirudin and one or more pharmaceutically acceptablestabilizing agents comprising a buffering agent having a pKa of about2.5 to about 6.5; for example, the buffering agent may be acetate. Thecomposition may further comprise a tonicity agent, such as sodiumchloride. In some embodiments, the composition may additionally comprisea preservative, such as methyl-paraben. In other embodiments, thecomposition may additionally comprise an antioxidant such as histidine,methionine, or a combination thereof.

In certain embodiments of the present invention, the pH may be adjustedby a pH-adjusting agent.

In embodiments of the present invention, the RTU bivalirudin compositioncomprises bivalirudin in an amount of about 5 mg/mL; a buffering agenthaving a pKa of about 2.5 to about 6.5, such as acetate;[9-10]-cycloimido bivalirudin; and [11-12]-cycloimido bivalirudin. Afterstorage at 25° C. for 1 month, total impurities may be less than about15% AUC, and, optionally, the [9-10]-cycloimido bivalirudin may bepresent in an amount greater than 0.2% AUC and less than about 5% AUC,and the [11-12]-cycloimido bivalirudin may be present in an amountgreater than 0.1% AUC and less than about 5% AUC. Further, thecomposition may have a pH of about 4.2.

In embodiments of the present invention, the RTU bivalirudin compositioncomprises bivalirudin in an amount of about 5 mg/mL; a buffering agenthaving a pKa of about 2.5 to about 6.5, such as acetate; sodiumchloride; [9-10]-cycloimido bivalirudin; and [11-12]-cycloimidobivalirudin. After storage at 25° C. for 1 month, total impurities maybe less than about 15% AUC, and, optionally, the [9-10]-cycloimidobivalirudin may be present in an amount greater than 0.2% AUC and lessthan about 5% AUC, and the [11-12]-cycloimido bivalirudin may be presentin an amount greater than 0.1% AUC and less than about 5% AUC. Further,the composition may have a pH of about 4.2.

The present invention additionally relates to a method of treating apatient in need thereof with an RTU bivalirudin composition. In variousembodiments, the method comprises administering an RTU bivalirudincomposition comprising bivalirudin and one or more stabilizing agents.In some embodiments, the RTU bivalirudin composition is an injectabledosage form, and can be delivered to the subject parenterally. Incertain embodiments, the RTU bivalirudin composition may be any of theRTU bivalirudin compositions described herein.

Furthermore, the present invention relates to a method of preparing anRTU bivalirudin composition comprising one or more stabilizing agents.In various embodiments, the method comprises mixing bivalirudin with oneor more stabilizing agents. The pH of the RTU bivalirudin compositionmay be adjusted by one or more pH-adjusting agents to obtain a pH ofabout 4 to less than 5. In some embodiments, the one or more stabilizingagents may be buffering agents having a pKa of about 2.5 to about 6.5,pH-adjusting agents, polymers, preservatives, antioxidants, sugars orpolyols, or a combination thereof.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE FIGURES

The following Detailed Description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings, in which:

FIG. 1 shows the likely degradation pathway for the conversion ofAsn-Gly residues into Asp-Gly residues;

FIG. 2 shows the likely degradation pathway for the conversion ofAsp-Phe residues into [11-12]-cycloimido bivalirudin;

FIG. 3 shows the mass spectrometry results for the analysis of[11-12]-cycloimido bivalirudin.

DETAILED DESCRIPTION

Peptides containing glutamine (“Gln”) and asparagine (“Asn”) residues,in general, are not stable in aqueous solution and are not suitable foran RTU product due to the many mechanisms that result in degradation ofthe peptide in solution. These peptides commonly undergo a type ofdegradation reaction known as deamidation at susceptible Gln and,especially, Asn residues. Deamidation can be catalyzed at neutral andalkaline pH via a succinimide intermediate, but can also occur at acidicpH via direct hydrolysis of the side-chain amide group of the Asnresidue. See Lindner et al., Exper. Geront. 36 (2001) 1551-1563 (“Linderet al.”). Consequently, it is difficult to prevent deamidation by simplepH adjustment. See generally WO 2009086062 at 1. Degradation viadeamidation is one of the reasons why most peptide-based pharmaceuticalsmust be produced in lyophilized (i.e., freeze-dried) form, requiringreconstitution before injection or administration. See id. at 2.

As depicted in FIG. 1, the Asn residues of peptides and proteins, inneutral or alkaline solutions, can be deamidated via a cyclicsuccinimide (“cycloimido”) intermediate with release of ammonia (“NH₃”).See Lindner et al., at 1552. The cycloimido intermediate, if it can bedetected at all, is usually present in only a very small amount. See id.at 1554. Hydrolysis of this intermediate can provide the correspondingpeptides containing L-aspartic acid (“L-Asp”) and L-isoaspartic acid(“L-isoAsp”). See id. at 1552. Moreover, racemization of theL-Asp-cycloimido peptide can lead to formation of the correspondingD-enantiomer, and after hydrolysis can lead to peptides containing D-Aspand D-isoAsp. See id. at 1553. Bischoff et al. describes the deamidationof recombinant hirudin in neutral and alkaline environments and suggeststhat hirudin undergoes the same deamidation mechanism as outlined inFIG. 1. See Bischoff et al., Biochemistry 32 (1993)725-734.

In acidic solutions, the mechanism of deamidation can be unpredictablesince some peptides can undergo deamidation by direct hydrolysis (seeid.) while other peptides can undergo deamidation by cycloimidointermediates (see Hekman et al., J. Pharm. Biomed. Anal., 20 (1999)763-772, and references therein).

Notably, some cycloimido intermediates appear to be unstable (seeLindner et al.) while others appear to be stable (see Hekman et al.); asa result, the stability of cycloimido intermediates can beunpredictable.

Bivalirudin undergoes degradation by pathways that are common to otherpeptides, including D-Phe-Pro cleavage (resulting in [3-20]-bivalirudin,SEQ ID NO: 2), deamidation, hydrolysis, and isomerization. Bivalirudinis susceptible to deamidation via the Asn⁹-Gly¹⁰ residues that can forma [9-10]-cycloimido bivalirudin intermediate (SEQ ID NO: 1, wherein Asn⁹and Gly¹⁰ form a succinimide) that can then undergo hydrolysis toAsp⁹-bivalirudin (SEQ ID NO: 3), isoAsp⁹-bivalirudin and isomersthereof. Asp⁹-bivalirudin is a known degradation and process impurity.See U.S. Patent Publication No. 20070093423 (“'423 publication”); U.S.application Ser. Nos. 12/180,550, 12/180,551, and 12/180,553.Controlling the formation of Asp⁹-bivalirudin has been an ongoingchallenge, although recent improvements in the preparation of Angiomax®have consistently controlled the formation of Asp⁹-bivalirudin generatedduring the compounding process. See U.S. application Ser. Nos.12/180,550; 12/180,551; and 12/180,553.

The development of RTU bivalirudin compositions of the present inventionrequired overcoming challenges regarding the stability of bivalirudin.Notably, Angiomax® is a lyophilized powder that requires reconstitutionprior to administration and has a pH of 5-6 (see Angiomax® PrescribingInformation). Once reconstituted, Angiomax® may be stored at 2-8° C. forup to 24 hours. Diluted Angiomax® may have a concentration of between0.5 mg/mL and 5 mg/mL is stable at room temperature for up to 24 hours.See id. Thus, reconstituted, or diluted, Angiomax® is not well suitedfor an RTU product requiring long term storage stability.

The inventors postulated that controlling Asp⁹-bivalirudin formationwould be the key challenge in developing a stable RTU bivalirudincomposition. Because of the unpredictability of both the deamidationmechanism and the stability of cycloimido intermediates, the inventorsconducted preliminary experiments investigating Asp⁹-bivalirudin andtotal impurity levels in bivalirudin solutions at a pH range of about 2to about 6. As described in Example 1, bivalirudin compositions wereprepared and monitored at time point zero and after storage at 25° C.for 1 month. At time point zero, the inventors observed that as pHincreased from about 2 to about 6 in the bivalirudin solutions, theminimum levels of Asp⁹-bivalirudin were observed at about pH 2 and 4,and the total impurities increased as the pH increased from about 2 toabout 6. After storage at 25° C. for 1 month, the bivalirudin solutionat about pH 4 had the least amount of Asp⁹-bivalirudin, althoughsignificant degradation of bivalirudin had occurred, presumably throughdegradation pathways other than the route depicted in FIG. 1. Theseresults appear to suggest that a pH of about 4 may aid in thestabilization of bivalirudin solutions as initially prepared, but pHalone was not likely the controlling factor in stabilizing bivalirudincompositions for long term storage.

The inventors discovered that the Asn⁹ residue of bivalirudin does notalways directly hydrolyze to Asp⁹ when at a pH of about 4 to less than 5(see, e.g., Lindner et al.). Rather, the Asn⁹-Gly¹⁰ residues can undergodeamidation to produce the [9-10]-cycloimido bivalirudin intermediate.The inventors also discovered that the [9-10]-cycloimido bivalirudinintermediate can be detectable, can accumulate, and may be isolatedunder certain conditions (e.g., 2-8° C.) without further degradation toAsp⁹-bivalirudin or isomers thereof. This can be observed in Examples39-42 wherein Asp⁹-bivalirudin levels (Imp. 4) increase at a slower ratethan its precursor, [9-10]-cycloimido bivalirudin (as contained in Imp.5), at 5° C. over 12 months.

The inventors discovered that maintaining the pH in a range of about 4to less than 5 can reduce the rate of bivalirudin deamidation into[9-10]-cycloimido bivalirudin and its further degradation intoAsp⁹-bivalirudin, thereby discovering a way of controlling the level ofAsp⁹-bivalirudin in solution. However, as mentioned above, pH alone isnot likely the controlling factor in stabilizing bivalirudincompositions for long term storage.

The inventors found that the concentration of other impurities increasedin bivalirudin solutions upon storage. The inventors determined thatthese impurities were formed by an alternative degradation mechanism andmust be controlled, in addition to Asp⁹-bivalirudin, in order to achievea stable RTU bivalirudin composition.

The inventors discovered that residues Asp¹¹ and phenylalanine-12(“Phe¹²”) surprisingly can form a succinimide intermediate that canaccumulate during storage in bivalirudin aqueous compositions at a pH ofabout 4 to less than 5. The formation of the intermediate would beunexpected given the large steric hindrance of the Phe residue.

As discovered by the inventors and shown in FIG. 2, the[11-12]-succinimide, herein called [11-12]-cycloimido bivalirudin (SEQID NO: 1, wherein the Asp¹¹ and Phe¹² form a succinimide), appears to bean intermediate of bivalirudin resulting from the cyclization of Asp¹¹and Phe¹² and subsequent loss of water. The inventors characterized the[11-12]-cycloimido bivalirudin by mass spectrometry. See FIG. 3. Theinventors contemplated that the [11-12]-cycloimido bivalirudin can behydrolyzed to bivalirudin isomers and peptide fragments including the[1-11]-bivalirudin (SEQ ID NO: 4) and [12-20]-bivalirudin (SEQ ID NO:5), and isomers thereof.

More surprisingly, the inventors found an accumulation of[11-12]-cycloimido bivalirudin (as contained in Imp. 6) in stable RTUbivalirudin compositions. Based on the bivalirudin purity in RTUbivalirudin compositions stored at 25° C. for 1 month, the inventorsbelieve that using one or more stabilizing agents minimized thehydrolysis of the [11-12]-cycloimido bivalirudin into the impuritiesidentified above. Thus, the stabilizing agents will likely slow downrate of formation and subsequent hydrolysis of [9-10]-cycloimidobivalirudin analogs resulting in enhanced bivalirudin stability.Likewise, the stabilizing agents will likely slow down rate of formationand subsequent hydrolysis of [11-12]-cycloimido bivalirudin analogsresulting in enhanced bivalirudin stability.

The present invention relates to an RTU bivalirudin compositioncomprising bivalirudin and one or more stabilizing agents. The presentinvention also relates to an RTU bivalirudin composition comprisingbivalirudin, one or more stabilizing agents, [9-10]-cycloimidobivalirudin, and [11-12]-cycloimido bivalirudin. The present inventionadditionally relates to a method of treating a patient in need thereofby administering an RTU bivalirudin composition comprising bivalirudinand one or more stabilizing agents. Further, the present inventionrelates to a method of preparing an RTU bivalirudin compositioncomprising bivalirudin and one or more stabilizing agents. The presentinvention also relates to a method of controlling the Asp⁹-bivalirudinlevels in a solution containing bivalirudin by slowing down the rate offormation of [9-10]-cycloimido bivalirudin and its subsequenthydrolysis.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. In the event that there is aplurality of definitions for a term used herein, those definitions inthis section prevail unless stated otherwise.

Definitions

As used herein, a “ready-to-use” or “RTU” composition is a sterile,aqueous or non-aqueous or a combination thereof, injectable compositionthat is stable and has not been reconstituted from a lyophilizate withinone day prior to use. The RTU composition is also a sterile, aqueous ornon-aqueous or a combination thereof, injectable composition that isstable and has been diluted from a concentrated, liquid solution.

As used herein, a “sterile” composition is one in which essentially allforms of microbial life have been destroyed by an appreciable amount tomeet the sterilization criteria outlined in the U.S. Pharmacopeia. SeeU.S. Pharmacopeia 32, NF 27, 1 (2009) 80-86.

As used herein, a “stable” composition does not exhibit appreciabledegradation upon storage over a set time limit, at a set temperature,and at an identified pH. In one embodiment, no more than about 15% ofbivalirudin is degraded upon storage at 25° C. over 1 month at a pH ofbetween about 4 to less than 5. In another embodiment, no more thanabout 12% of bivalirudin is degraded upon storage at a temperature ofabout 2° C. to about 8° C. over 12 months at a pH of between about 4 toless than 5. In another embodiment, a single impurity does not exceedabout 5% after storage at 25° C. for 1 month.

A “stabilizing agent” is any component that allows bivalirudin to bestable.

Various analytical techniques for measuring protein stability areavailable in the art and are reviewed in Lee, Peptide and Protein DrugDelivery (1991) 247-301, Marcel Dekker, Inc., New York, N.Y. and Jones,Adv. Drug Delivery Rev. 10 (1993) 29-90. Typically, a high performanceliquid chromatography (“HPLC”) system with appropriate hardware,software, solvents and reference standards is employed for the analysisof impurities.

As used herein, the term “has not been reconstituted from alyophilizate” means that a solid has not been dissolved or suspended.

Bivalirudin

Bivalirudin can be synthesized by methods that include, but are notlimited to, solid-phase peptide synthesis, solution-phase peptidesynthesis, or a combination of solid-phase and solution-phase procedures(see, e.g., '404 patent; Okayama et al., Chem. Pharm. Bull., 44 (1996)1344-1350; Steinmetzer et al., Eur. J. Biochem., 265 (1999) 598-605;'423 publication; U.S. Patent Publication No. 2008051558; U.S. PatentPublication No. 2008287648).

The bivalirudin in the RTU compositions may be the peptide encoded bySEQ ID NO: 1, or salts thereof. Bivalirudin may be present in an amountcomprising between about 0.01 mg/mL and about 100 mg/mL, or betweenabout 0.05 mg/mL and about 50 mg/mL, or between about 0.1 mg/mL andabout 25 mg/mL, or between about 1.0 mg/mL and about 10 mg/mL, orbetween about 2.5 mg/mL and 7.5 mg/mL, such as a concentration of about5.0 mg/mL.

Cycloimido-Bivalirudin and Bivalirudin Fragments

The RTU bivalirudin compositions of the present invention may compriseone or more cycloimido-bivalirudins and/or bivalirudin fragments. Forexample, RTU bivalirudin compositions may comprise [9-10]-cycloimidobivalirudin, [11-12]-cycloimido bivalirudin, or a combination thereof.After storage at 25° C. for 1 month, [9-10]-cycloimido bivalirudin maybe greater than 0.2% AUC to less than about 5% AUC, or may be greaterthan about 0.5% AUC to less than about 3% AUC, while the[11-12]-cycloimido bivalirudin may be greater than 0.1% AUC to less thanabout 5% AUC, or may be greater than about 1% AUC to less than about 4%AUC, or may be greater than about 2% AUC to less than about 3% AUC.

Similarly, after storage at 5° C. for 12 months, [9-10]-cycloimidobivalirudin may be greater than 0.2% AUC to less than about 5% AUC, ormay be greater than about 0.5% AUC to less than about 3% AUC, and the[11-12]-cycloimido bivalirudin may be greater than 0.1% AUC to less thanabout 5% AUC, or may be greater than about 1% AUC to less than about 4%AUC, or may be greater than about 2% AUC to less than about 3% AUC.

RTU bivalirudin compositions may also comprise one or more bivalirudinfragments, such as [3-20]-bivalirudin (SEQ ID NO: 2), [1-11]-bivalirudin(SEQ ID NO: 4), [12-20]-bivalirudin (SEQ ID NO: 5), or a combinationthereof.

Stabilizing Agents

The compositions of the present invention may comprise one or morepharmaceutically acceptable stabilizing agents that include, but are notlimited to, one or more buffering agents having a pKa of about 2.5 toabout 6.5, pH-adjusting agents, polymers, preservatives, antioxidants,sugars or polyols, or a combination thereof. In various embodiments, thecompositions further comprise a tonicity adjusting agent.

Buffering agents may comprise pharmaceutically acceptable reagents orcomponents that contribute to maintaining the pH of bivalirudincompositions between about 4 to about less than 5. Such buffering agentsmay typically have pKa values that are within the target pH of thebivalirudin compositions, plus or minus about one and a half pH units.For example, buffering agents that have a pKa of about 2.5 to about 6.5are included within the scope of the invention. Furthermore, mixedbuffers wherein one buffer component falls within the pKa range of about2.5 to about 6.5 are included within the scope of the invention. Suchbuffering agents that are included within the scope of the inventioninclude, but are not limited to, ascorbate, lactobionate, gentisate,succinate, α-lipoic acid, maleate, chloroacetate, citrate, bicarbonate,tartrate, glycylglycine, formate, benzoate, citrate, lactate, acetate,propionate, pyridine, piperazine, pyrophosphate, histidine,2-(N-morpholino)ethanesulfonic acid (“MES”), cacodylic acid,(bis(2-hydroxyethyl)-imino-tris(hydroxymethyl)-methane) (“bis-TRIS”),bicarbonate, or a combination of these buffering agents.

In one embodiment, the buffering agent may comprise a buffer having apKa value between about 3.5 and about 5, which may include, but is notlimited to, formate, benzoate, citrate, acetate, tartrate and propionatebuffering agents. In particular embodiments, the buffering agent maycomprise a buffer having a pKa of about 4.2 to about 4.5, such astartrate or acetate buffering agents.

In various embodiments, the concentration of the buffering agents in thecomposition may be between about 0.01 M and about 10 M. In variousembodiments, the buffering agent is present in a concentration ofbetween about 0.1 M and 1 M.

The pH-adjusting agents are pharmaceutically acceptable components orreagents that are used to adjust the final pH of the RTU bivalirudincomposition, the pH during the preparation of the RTU bivalirudincomposition, and the pH of the buffering agent. For example, during thepreparation of an RTU bivalirudin composition of the present invention,bivalirudin could be added to a buffering agent resulting in a change inthe pH of the buffer solution. A pH-adjusting agent could be added toadjust the pH to the desired level during the addition of bivalirudin tothe buffering agent, or after all the bivalirudin has been added. ThepH-adjusting agents may include pharmaceutically acceptable acids,bases, or buffering agents. For example, the acids may include, but arenot limited to, one or more inorganic mineral acids such ashydrochloric, hydrobromic, sulfuric, phosphoric, nitric, and the like;or one or more organic acids such as acetic, succinic, tartaric,ascorbic, citric, glutamic, benzoic, methanesulphonic, ethanesulfonic,trifluoroacetic and the like. The bases may be one or more inorganicbases or organic bases, including, but not limited to, alkalinecarbonate, alkaline bicarbonate, alkaline earth metal carbonate,alkaline hydroxide, alkaline earth metal hydroxide or amine. Forexample, the inorganic or organic base may be an alkaline hydroxide suchas lithium hydroxide, potassium hydroxide, cesium hydroxide, sodiumhydroxide or the like; an alkaline carbonate such as calcium carbonate,sodium carbonate or the like; or an alkaline bicarbonate such as sodiumbicarbonate or the like; the organic base may also be sodium acetate.Examples of buffering agents are described above.

Pharmaceutically acceptable polymers may comprise, but are not limitedto, fatty esters of polyalcohols, polyalkyleneglycols and mixedpolyalkyleneglycol copolymers such as poloxamer, polyethers,polyoxyethylenated fatty alcohols and esters, polysorbates (“Tween”),polyvinyl alcohols, polyalkylene oxides, polyacrylamides,polyvinylpyrrolidones, hydroxyethyl starch or combinations thereof. Incertain embodiments, the polymer may be polyethyleneglycol (“PEG”).Polymers may be present in a concentration of between about 1% and about50%, or between about 5% and about 25%, or between about 10% and about20%.

Preservatives may comprise, but are not limited to, benzalkoniumchloride, bronopol, cetrimide (“cetyltrimethylammonium bromide”),benzoic acid, benzyl alcohol, borates, chlorhexidine, chlorobutanol,nitrates, alkyl parabens including methyl- and ethyl- andpropyl-paraben, phenylmercuric acetate, potassium sorbate, sodiumbenzoate, sorbic acid, thiomersal (“mercurithiosalicylate”), orcombinations thereof. Preservatives may be present in the compositionsin an amount of between about 0.01% w/w and about 5% w/w, or about 0.05%w/w and about 3% w/w, or about 0.1% w/w and about 1% w/w, or about 0.1%w/w and about 0.5% w/w, or about 0.2% w/w.

Antioxidants may comprise, but are not limited to, acetylcysteine,ascorbyl palmitate, butylated hydroxyanisole (“BHA”), butylatedhydroxytoluene (“BHT”), monothioglycerol, potassium nitrate, sodiumascorbate, sodium formaldehyde sulfoxylate, sodium metabisulfite, sodiumbisulfite, vitamin E or a derivative thereof, propyl gallate, edetate(“EDTA”) (e.g., disodium edetate), diethylenetriaminepentaacetic acid(“DTPA”), triglycollamate (“NT”), or a combination thereof. Antioxidantsmay also comprise amino acids such as methionine, histidine, cysteineand those carrying a charged side chain, such as arginine, lysine,aspartic acid, and glutamic acid. Any stereoisomer (e.g., L-, D-, or acombination thereof) of any particular amino acid (e.g., methionine,histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan,threonine and combinations thereof) or combinations of thesestereoisomers, may be present so long as the amino acid is presenteither in its free base form or its salt form. For example, theL-stereoisomer is used.

In further embodiments, antioxidants can also include analogues of aminoacids. The term “amino acid analogue” encompasses a derivative of thenaturally occurring amino acid that brings about the desired effect ofstabilizing the cycloimido bivalirudins and decreasing aggregateformation by the peptide during storage of the RTU bivalirudincompositions of the present invention. Suitable amino acid analoguesinclude, but are not limited to, arginine analogues such asaminoguanidine, ornithine and N-monoethyl L-arginine; methionineanalogues such as ethionine and buthionine; and suitable cysteineanalogues such as S-methyl-L-cysteine. The amino acid analogues can beincorporated into the RTU compositions in either their free base form ortheir salt form.

Antioxidants may be present in the compositions in a quantity of betweenabout 0.01% w/w and about 1% w/w, or about 0.01% w/w and about 0.5% w/w,or about 0.01% w/w and about 0.3% w/w, or about 0.01% w/w and about 0.1%w/w.

Sugars or polyols may comprise, but are not limited to, glycerin,sucrose, lactose, glucose, fructose, arabinose, xylose, ribose, mannose,galactose, dextrose, sorbose, sorbitol, mannitol, maltose, cellobiose,xylitol, or combinations thereof. The sugars or polyols may be presentin the compositions in a quantity of about 1% to about 10%.

Tonicity adjusting agents, as used here, are agents that adjust the RTUbivalirudin composition to the desired isotonic range. The term“isotonic” means “isotonic with serum”. An acceptable range is about 200to about 1000 mOsm/kg. In various embodiments, the tonicity of the RTUbivalirudin composition is between about 200 to about 600 mOsm/kg. Thetonicity agents that may be used in the RTU compositions may include,but are not limited to, pharmaceutically acceptable inorganic chloridessuch as sodium chloride, potassium chloride, magnesium chloride, orcalcium chloride; sugars such as dextrose, glycerol, lactose, sucrose,mannitol, sorbitol, and the like; or combinations thereof. Certaintonicity adjusting agents have been found to enhance the stability ofRTU bivalirudin compositions containing other stabilizing agents.

The quantity of tonicity agent in the composition may vary according tothe agent and according to the desired isotonic range. For example, thetonicity agent may be present in an amount between about 1 mg/mL andabout 25 mg/mL, or between about 5 mg/mL and 20 mg/mL, or about 7 mg/mLand about 10 mg/mL. In certain embodiments, tonicity agent may be sodiumchloride at a quantity of about 9 mg/mL, or dextrose at a quantity ofabout 50 mg/mL. In various embodiments, certain tonicity agents may alsoserve as the stabilizing agent.

RTU bivalirudin compositions of the present invention may furthercomprise a salt of a pH adjusting agent. During the preparation of thecompositions, a pH adjusting agent may be required to obtain the targetpH of the RTU bivalirudin composition. The pH adjusting agents mayinclude pharmaceutically acceptable acids, bases, or buffering agents.Hence, the composition may include salts of any of the one or moreacids, bases, or buffering agents.

Also, the pH of the final RTU bivalirudin composition of the presentinvention is between about 4 to less than 5. The hydrogen ionconcentration is about 1×10⁻⁴ M to less than about 1×10⁻⁵ M.

Notably, the inventors surprisingly found that RTU bivalirudincompositions comprising different stabilizing agents, in some cases, hadthe same largest individual impurity at low temperature (i.e., 5° C.),while it was different at a higher temperature (i.e., 25° C.). Theinventors contemplated that some stabilizing agents stabilize differentdegradation mechanisms at different temperatures.

Arrhenius Kinetics

Arrhenius kinetics can be used to predict the shelf life of RTUbivalirudin compositions at 5° C., according to the equation:

k _(obs) =Ae ^(−Ea/RT)

wherein, k_(obs) is the observed rate constant, A is the pre-exponentialfactor, Ea is the activation energy and R is the gas constant. Accordingto Example 43, RTU bivalirudin compositions of Examples 32-37 and 39-40are stable for at least 16 months of storage at 5° C.

Method of Treatment

The present invention relates to a method of treating a patient in needthereof. The method comprises administering an RTU bivalirudincomposition comprising bivalirudin and one or more stabilizing agents.The RTU bivalirudin composition may be any RTU composition describedherein.

The RTU bivalirudin composition can be an injectable dosage form, andcan be delivered to the subject parenterally. Methods of delivering theRTU bivalirudin composition parenterally are well known in the art. Forexample, the aqueous composition may be delivered intravenously.

The aqueous composition may be an intravenous bolus dose of betweenabout 0.25 mg/kg and about 1.5 mg/kg, or between about 0.5 mg/kg toabout 1 mg/kg, or about 0.75 mg/kg. This may be followed by an infusionof between about 1.25 mg/kg/h and about 2.25 mg/kg/h, or about 1.75mg/kg/h for the duration of the procedure or treatment protocol. Fiveminutes after the bolus dose is administered, an additional bolus ofbetween about 0.1 mg/kg and about 1 mg/kg, or about 0.3 mg/kg, may begiven if needed.

The RTU bivalirudin composition of the present invention can beindicated for use as an anticoagulant. Also, the RTU bivalirudincomposition can be used for the prevention and treatment of venousthromboembolic disease. Likely indications include treatment in patientswith unstable angina undergoing percutaneous transluminal coronaryangioplasty; administration with the provisional use of glycoproteinIIb/IIIa inhibitor for use as an anticoagulant in patients undergoingPCI; and treatment in patients with, or at risk of, HIT or HITTSundergoing PCI. Also, RTU bivalirudin composition can be used for theprevention and treatment of venous thromboembolic disease.

The RTU bivalirudin composition may be administered with other drugproducts such as glycoprotein IIb/IIIa inhibitor (see, e.g., Allie etal., Vasc. Dis. Manage. 3 (2006) 368-375). Alternatively, RTUbivalirudin compositions may be combined with blood thinners including,but not limited to, coumadin, warfarin, and preferably, aspirin.

Method of Preparing a RTU Bivalirudin Composition

The present invention relates to a method of preparing an RTUbivalirudin composition comprising bivalirudin and one or morestabilizing agents. The method comprises mixing one or more stabilizingagents with bivalirudin to form the RTU bivalirudin composition.Optionally, the method may also comprise adjusting the pH of the RTUbivalirudin composition if the solution does not have a pH of about 4 toless than 5.

The one or more stabilizing agents may first be dissolved in water priorto mixing with bivalirudin. The stabilizing agents may comprise any ofthe buffering agents, pH-adjusting agents, polymers, preservatives,antioxidants, sugars or polyols, or a combination thereof, as describedabove.

The one or more stabilizing agents may be dissolved in water by methodsknown in the art. For example, the stabilizing agents may be dissolvedby adding each stabilizing agent to water, by adding the stabilizingagents to each other and then mixing them with water, by mixing the oneor more stabilizing agents and water in a common receptacle, or acombination thereof. The one or more stabilizing agents may be addedsimultaneously, individually in a particular order, individually in anyorder, or a combination thereof, e.g., some stabilizing agents are addedtogether while others are added individually in a particular order.Reasons for adding stabilizing agents in a particular order may include,but are not limited to, preventing a reaction that may occur when twoparticular agents are mixed directly together, stimulating a reactionthat may occur when two particular agents are mixed directly together,maintaining a certain pH, tonicity, etc., and ease of handling.

The one or more stabilizing agents may be dissolved in water using amixing device that is known in the art. Examples of mixing devices mayinclude, but are not limited to, a paddle mixer, magnetic stirrer,shaker, re-circulating pump, homogenizer, and any combination thereof.The mixing device may be applied at a mixing rate between about 10 andabout 1500 rpm, and for between about 0.1 and about 120 minutes. Themixing device may be applied constantly as one or more stabilizingagents are added to water, sporadically, or a combination thereof, e.g.,certain stabilizing agents may require more mixing than others.

The dissolution of the one or more stabilizing agents may occur undercontrolled conditions. For example, temperature may be controlled bymeans known in the art, such as by mixing the stabilizing agentstogether in a vessel inside a cooling jacket. The temperature may be setbetween about 1° C. and about 25° C., or between about 2° C. and about10° C. Also, the dissolution of the one or more stabilizing agents mayoccur under conditions such as under nitrogen or at a particularhumidity, etc.

Thereafter, the bivalirudin may be mixed with the dissolved stabilizingagents by methods known in the art. Bivalirudin may be added to thedissolved stabilizing agents rapidly, slowly, all at once, in portions,at a constant rate, at a variable rate, or a combination thereof.Alternatively, the stabilizing solution may be added to bivalirudin, orthe stabilizing solution and bivalirudin may be added together into acommon receptacle.

A mixing device known in the art may be used to mix bivalirudin and thedissolved stabilizing agents. Examples of mixing devices may include,but are not limited to, a paddle mixer, magnetic stirrer, shaker,re-circulating pump, homogenizer, and any combination thereof. Themixing device may be applied at a mixing rate between about 10 and about1500 rpm, and for between about 0.1 and about 120 minutes. Whetherbivalirudin is added to the dissolved stabilizing agents or vice versa,or whether they are added simultaneously into a common receptacle, themixing device may be applied constantly, or sporadically, or acombination thereof, as the mixing proceeds.

The mixing of bivalirudin with the dissolved stabilizing agents mayoccur under controlled conditions. For example, temperature may becontrolled by means known in the art, such as by mixing the bivalirudinand the dissolved stabilizing agents together in a vessel inside acooling jacket. The temperature may be set between about 1° C. and about25° C., or between about 2° C. and about 10° C. Also, the mixing ofbivalirudin and the dissolved stabilizing agents may occur underconditions such as under nitrogen or at a particular humidity, etc.

The bivalirudin may already by dissolved in water when it is mixed withthe dissolved stabilizing agents. On the other hand, the bivalirudin maybe mixed in its solid form with the dissolved stabilizing agents.

In an alternative embodiment, bivalirudin may be mixed with one or morestabilizing agents prior to dissolution in water, simultaneously withdissolution in water, or a combination thereof. For example, solidbivalirudin and a first stabilizing agent may be mixed together and thendissolved in water, and then a second stabilizing agent may be mixedwith the dissolved bivalirudin and first stabilizing agent. As anotherexample, a first stabilizing agent may be dissolved in water and thenmixed with bivalirudin (either in solid form or dissolved), and thisresulting solution may be mixed with a second stabilizing agent(dissolved or in solid form). Such various orders of addition and mixingare all embodiments of the present invention. Notably, each of thesemixing methods may include the use of mixing devices or particularmixing conditions as described above.

The solution comprising the dissolved bivalirudin and stabilizing agentsis an RTU bivalirudin composition as described in certain embodiments ofthe present invention.

In certain embodiments of the present invention, a desired pH for theRTU bivalirudin composition may be about 4 to less than 5. If the pH ofthe RTU bivalirudin composition is not in this range after performingthe method disclosed above, one or more pH-adjusting agents may be addedto the RTU bivalirudin composition to achieve the desired pH.

Moreover, if a specific concentration of bivalirudin is desired in theRTU bivalirudin composition, the concentration can be adjusted, forexample, by the addition of water to the RTU bivalirudin composition.

After the RTU bivalirudin composition is prepared, it may be sterilized.For instance, the aqueous composition may undergo aseptic filtrationusing, for example, a 0.2 μm disposable membrane filter. Also,sterilization may involve a freeze-thaw cycle to kill any residualvegetative bacteria. Techniques of sterilizing the aqueous compositionare known in the art. See, e.g., Berovic, Biotechnol. Annu. Rev. 11(2005) 257-79.

The RTU bivalirudin composition may be placed into a container having asterile access port for piercing by a hypodermic injection needle, forexample, an intravenous solution bag, bottle, vial, ampoule, pre-filledsterile syringe, together with instructions for administration.

Method of Controlling Asp⁹-Bivalirudin Levels

The present invention also relates to a method of controlling theAsp⁹-bivalirudin levels in a solution containing bivalirudin. Thismethod may comprise mixing the solution with one or more stabilizingagents as described above, slowing down the rate of formation of[9-10]-cycloimido bivalirudin and its subsequent hydrolysis, therebycontrolling Asp⁹-bivalirudin levels and stabilizing bivalirudin.

This method can be used to control Asp⁹-bivalirudin levels in solutionscontaining bivalirudin that are stored, for example, at 25° C. for onemonth, or at 5° C. for 12 months.

The invention will now be further described by way of the followingnon-limiting examples, which further illustrate the invention; suchexamples are not intended, nor should they be interpreted, to limit thescope of the invention.

Examples

The examples reported herein below illustrate the preferred embodimentsof the present invention in greater detail but should not be construedto limit the invention in any way.

Example 1 Investigation of pH of Bivalirudin Solutions

The degradation pathway of bivalirudin was investigated by preparingaqueous solutions at 1 mg/ml of bivalirudin at pH 2, 3, 4, 5 and 6(Table 1). Notably, the pH of a 1 mg/mL bivalirudin aqueous solution isabout 2.8; thus aqueous solutions of sodium hydroxide or trifluoroaceticacid were added to adjust the pH to the desired level. At the initialtime point, the inventors observed that as the pH was increased fromabout 2 to about 6, the total degradants also increased. After 1 monthof storage at 25° C., significant potency loss was observed for allbivalirudin samples. Bivalirudin compositions exhibited better stabilitybetween pH of about 3 and about 4. The excessive degradation observedwas attributed to a change in pH during storage wherein the final pH ofthe bivalirudin compositions was about 7.

TABLE 1 Asp⁹-Bivalirudin Total Degradants pH Time (% AUC) (% AUC) 2Initial 0.45 0.6 3 Initial 0.76 0.9 4 Initial 0.59 3.1 5 Initial 0.843.4 6 Initial 1.19 4.7 2 25° C./1 month 2.58 64.8 3 25° C./1 month 2.7137.7 4 25° C./1 month 1.26 51.1 5 25° C./1 month complete degradation 625° C./1 month complete degradation

Example 2 Investigation of Various Buffering Agents Added to BivalirudinSolutions

Various buffering agents were investigated to stabilize the pH of thebivalirudin aqueous solutions. Buffered solutions of bivalirudin wereprepared at pH 4.0, 4.5, 5.0, and 5.5 and the stability resultsindicated improved stability at a pH of about 4 to less than 5.

This result was surprising as the current Angiomax® product has a pH ofabout 5.2 following reconstitution prior to injection. Various bufferingagents are contemplated within the scope of the invention that have anadequate buffering capacity in the pH range of about pH 4 to about lessthan 5, and a pKa range of about 2.5 to about 6.5.

Example 3 General Procedure for the Preparation of Laboratory Scale RTUBivalirudin Compositions

A 1 M acetic acid solution was prepared by diluting 11.5 mL of aceticacid with water in a 200 mL volumetric flask. A 1 N sodium hydroxide(“NaOH”) solution was prepared by dissolving 4 g of NaOH with water in a100 mL volumetric flask. A 0.05 M sodium acetate solution was preparedby dissolving 0.68 g of sodium acetate trihydrate in 50 mL of water.

To the sodium acetate solution was optionally added stabilizing agents(e.g., 5 mL of PEG 400). The pH of the sodium acetate solution was thenadjusted to between about 4 to less than 5 with the addition of 1 Macetic acid solution.

Bivalirudin (100 mg) was slowly added with stirring to 10 mL of thesodium acetate buffer solution. The pH was adjusted to between about 4to less than 5 with the addition of the 1 N NaOH solution and water wasadded (q.s. to 20 mL). The solution was filtered through a 0.45 μmmembrane filter into USP Type I flint glass vials, stoppered withbromobutyl stoppers and sealed with aluminum caps.

Example 4 General HPLC Method for Analysis of Laboratory Scale RTUBivalirudin Compositions

Storage stability assessment was performed on RTU bivalirudincompositions of Examples 5-20 by means of HPLC using the followingexperimental conditions:

-   Mobile Phase A: 0.1% TFA in water-   Mobile Phase B: 0.1% TFA in acetonitrile-   Column: Jupiter Proteo 90 A°, 4 μm Column, 2.0 mm×250 mm-   Pump Mode: Gradient-   Flow Rate: 0.23 mL/min-   Wavelength: 215 nm-   Sample Cooler: 15° C.-   Injection volume 5 μL-   Run time: 65 minutes-   Column temperature: 45° C.-   Gradient Program: see TABLE 2

TABLE 2 Mobile Phase B Time % 0.01 5 5.00 5 10.10 22 35.00 27 40.00 10050.00 100 51.00 5 65.00 5 65.01 StopFollowing these conditions, the following impurities were observed:

TABLE 3 No. Name RRT Imp. 1 [1-11]-bivalirudin 0.55 Imp. 2[12-20]-bivalirudin 0.82 Imp. 3 [3-20]-bivalirudin 0.85 Imp. 4Asp⁹-bivalirudin 1.04 Imp. 5 Co-eluting peaks that include 1.08[9-10]-cycloimido-bivalirudin Imp. 6 Co-eluting peaks that include 1.18[11-12]-cycloimido-bivalirudinDepending on the RTU bivalirudin composition, the relative retentiontime (“RRT”) values can shift by about 0.1 RRT units.

Example 5

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 4 was prepared.

TABLE 4 Ingredients Quantity Bivalirudin 10 mg/mL Sodium acetate 0.05 MSodium hydroxide 1 M q.s. to pH 4.5 Water q.s.

The samples were stored at 25° C. for 1 month and the stability data ispresented below in Table 5.

TABLE 5 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0 0 0 0 0 0.13 0.13 25° C./1 month 0.28 2.17 0.97 0.51 1.07 1.24 7.4

Example 6

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 6 was prepared.

TABLE 6 Ingredients Quantity Bivalirudin 5 mg/mL Sodium acetate 0.05 MPEG 400 5% Sodium hydroxide 1 M q.s. to pH 4 Water q.s.

The samples were stored at 25° C. for 1 month and the stability data ispresented below in Table 7.

TABLE 7 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0 0 0 0 0 0 0 25° C. /1month 0.42 1.38 1.00 0.56 0.57 2.10 7.3

Example 7

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 8 was prepared.

TABLE 8 Ingredients Quantity Bivalirudin 5 mg/mL Sodium acetate 0.05 MSodium hydroxide 1 M q.s. to pH 4 Water q.s.

The samples were stored at 25° C. for 1 month and the stability data ispresented below in Table 9.

TABLE 9 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0 0 0 0 0 0 0 25° C. /1month 0 1.38 1.07 0.57 0.54 1.92 6.5

Example 8

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 10 was prepared.

TABLE 10 Ingredients Quantity Bivalirudin 5 mg/mL L-Histidine 0.05 MSodium acetate 0.01 M Acetic acid 1 M q.s. to pH 4.5 Water q.s.

The samples were stored at 25° C. for 1 month and 5° C. for 8 months andthe stability data is presented below in Table 11.

TABLE 11 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0 0 0.04 0.21 0.04 0 0.425° C. /1 month 0.74 2.34 0.97 1.18 1.14 2.15 8.8 5° C. /8 months 0.441.77 0.94 1.47 1.09 2.28 10.0

Example 9

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 12 was prepared.

TABLE 12 Ingredients Quantity Bivalirudin 5 mg/mL Sodium acetate 0.05 MLactobionic acid 2 mg/mL HES 5% Acetic acid 2 M q.s. to pH 4.5 Waterq.s.

The samples were stored at 25° C. for 1 month and the stability data ispresented below in Table 13.

TABLE 13 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0 0 0 0.26 0 0 0.4 25° C./1 month 0.80 2.40 0.91 1.15 1.13 0.54 7.6

Example 10

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 14 was prepared.

TABLE 14 Ingredients Quantity Bivalirudin 5 mg/mL Sodium acetate 0.05 MHES 50 mg/mL Sucrose 2 mg/mL Acetic acid 2 M q.s. to pH 4.25 Water q.s.

The samples were stored at 25° C. for 1 month, 5° C. for 8 months, and5° C. for 11 months. The stability data is presented below in Table 15.

TABLE 15 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0 0 0 0.19 0 0 0.3 25° C./1 month 0.59 1.06 0 1.11 1.07 2.08 9.3 5° C. /8 months 0.35 1.90 1.141.50 1.24 2.47 10.0 5° C. /11 months 0.10 1.61 1.00 1.67 1.13 3.58 10.6

Example 11

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 16 was prepared.

TABLE 16 Ingredients Quantity Bivalirudin 5 mg/mL Sodium acetate 0.05 MHydrochloric acid q.s. to pH 4.5 Sodium hydroxide 1 M q.s. to pH 4.5Water q.s.

The samples were stored at 25° C. for 1 month and the stability data ispresented below in Table 17.

TABLE 17 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0 0 0 0.28 0 0 0.5 25°C./1 month 0.31 1.09 0.80 0.65 0.70 1.14 5.3

Example 12

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 18 was prepared.

TABLE 18 Ingredients Quantity Bivalirudin 5 mg/mL Succinic acid 0.05 MSodium hydroxide 1 M q.s. to pH 4 Water q.s.

The samples were stored at 25° C. for 1 month and the stability data ispresented below in Table 19.

TABLE 19 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0 0 0.38 0.22 0 0 0.9 25°C./1 month 0.19 0.74 1.37 0.50 0.26 1.13 5.4

Example 13

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 20 was prepared.

TABLE 20 Ingredients Quantity Bivalirudin 5 mg/mL Succinic acid 0.05 MSodium hydroxide 1 M q.s. to pH 4.5 Water q.s.

The samples were stored at 25° C. for 1 month and the stability data ispresented below in Table 21.

TABLE 21 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0 0 0.31 0.63 0 0 1.3 25°C./1 month 0.35 1.23 0.94 0.73 0.59 1.74 7.1

Example 14

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 22 was prepared.

TABLE 22 Ingredients Quantity Bivalirudin 5 mg/mL Sodium citrate 0.05 MHydrochloric acid q.s. to pH 4 Sodium hydroxide 1 M q.s. to pH 4 Waterq.s.

The samples were stored at 25° C. for 1 month and the stability data ispresented below in Table 23.

TABLE 23 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0 0 0.31 1.10 0 0 1.6 25°C./1 month 0.19 0.77 1.55 0.66 0.46 2.32 6.8

Example 15

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 24 was prepared.

TABLE 24 Ingredients Quantity Bivalirudin 5 mg/mL Lactic acid 0.5 MSodium hydroxide 1 M q.s. to pH 4 Water q.s.

The samples were stored at 25° C. for 1 month and the stability data ispresented below in Table 25.

TABLE 25 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0 0 0.24 0.26 0 0 0.7 25°C./1 month 0.37 0.97 1.27 0.73 0.38 1.66 7.7

Example 16

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 26 was prepared.

TABLE 26 Ingredients Quantity Bivalirudin 5 mg/mL Lactic acid 0.5 MSodium hydroxide 1 M q.s. to pH 4.5 Water q.s.

The samples were stored at 25° C. for 1 month and the stability data ispresented below in Table 27.

TABLE 27 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0 0 0.26 0.62 0.94 0 2.025° C./1 month 0.32 1.23 0.83 0.58 0.50 1.53 5.3

Example 17

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 28 was prepared.

TABLE 28 Ingredients Quantity Bivalirudin 5 mg/mL Tartaric acid 0.05 MSodium hydroxide 1 M q.s. to pH 4 Water q.s.

The samples were stored at 25° C. for 1 month and the stability data ispresented below in Table 29.

TABLE 29 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0 0 0.16 0.23 0 0 0.6 25°C./1 month 0.14 0.67 1.32 0.54 0.34 1.80 5.8

Example 18

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 30 was prepared.

TABLE 30 Ingredients Quantity Bivalirudin 5 mg/mL Tartaric acid 0.05 MSodium hydroxide 1 M q.s. to pH 4.5 Water q.s.

The samples were stored at 25° C. for 1 month and the stability data ispresented below in Table 31.

TABLE 31 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0 0 0.20 0.16 0 0 0.6 25°C./1 month 0.24 1.05 0.57 0.92 0.79 0.64 6.1

Example 19

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 32 was prepared.

TABLE 32 Ingredients Quantity Bivalirudin 50 mg/mL α-Lipoic acid 25μg/mL Sucrose 5% Sodium hydroxide 1 M q.s. to pH 4.5 Water q.s.

The samples were stored at 25° C. for 1 month and the stability data ispresented below in Table 33.

TABLE 33 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0 0 0 0.62 0 0 0.9 25°C./1 month 0.83 0.90 0.79 1.29 0.73 1.49 7.3

Example 20

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 34 was prepared.

TABLE 34 Ingredients Quantity Bivalirudin 50 mg/mL Sucrose  5% HES 10%Sodium hydroxide 1 M q.s. to pH 4.5 Water q.s.

The samples were stored at 25° C. for 1 month and the stability data ispresented below in Table 35.

TABLE 35 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0 0 0.32 0.46 0 0.09 1.325° C./1 month 0.30 0.95 0.61 0.68 1.42 1.06 6.3

Example 21 Alternative HPLC Method for Analysis of RTU BivalirudinCompositions

Storage stability assessment was performed on RTU bivalirudincompositions of Examples 22-37, 39-42 by means of HPLC using thefollowing experimental conditions:

-   Mobile Phase A: sodium acetate (50 mM, pH 6.6)-   Mobile Phase B: 50% acetonitrile: 50% sodium acetate (50 mM, pH 6.6)-   Column: Vydac C18, 5 μm, 250 mm×4.6 mm-   Pump Mode: Gradient-   Flow Rate: 1.2 mL/min-   Wavelength: 215 nm-   Injection volume: 40 μL-   Run time: 40 minutes-   Column temperature: 40° C.-   Gradient Program: see TABLE 36

TABLE 36 Mobile Phase B Time % 0 10 5 15 30 35 35 35 35.01 10 40 10Following these conditions, the following impurities were observed:

TABLE 37 No. Name RRT Imp. 1 [1-11]-bivalirudin 0.49 Imp. 2[12-20]-bivalirudin 0.57 Imp. 3 [3-20]-bivalirudin 0.62 Imp. 4Asp⁹-bivalirudin 0.90 Imp. 5 Co-eluting peaks that include 1.07[9-10]-cycloimido-bivalirudin Imp. 6 Co-eluting peaks that include 1.36[11-12]-cycloimido-bivalirudinDepending on the RTU bivalirudin composition, the relative retentiontime (“RRT”) values can shift by about 0.1 RRT units.

Example 22

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 38 was prepared.

TABLE 38 Ingredients Quantity Bivalirudin 5 mg/mL Sodium acetate 50 mMSodium hydroxide 1 M q.s. to pH 4.5 Water q.s.

The samples were stored at 40° C. for 3 and 7 days and the stabilitydata is presented below in Table 39.

TABLE 39 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial NR 0.32 0.08 0.47 0.150.06 1.6 40° C./3 days NR 1.08 1.61 0.45 0.92 2.12 7.7 40° C./7 days NR2.08 3.17 0.61 1.59 3.72 14.0 NR = not recorded

Example 23

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 40 was prepared.

TABLE 40 Ingredients Quantity Bivalirudin 5 mg/mL Sodium acetate 50 mMGlycine 0.5%  Tween 80 0.02%   Sucrose 10% Glycerol 10% Sodium hydroxide1 M q.s. to pH 4.5 Water q.s.

The samples were stored at 40° C. for 3 and 7 days and the stabilitydata is presented below in Table 41.

TABLE 41 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.radants Time 1 2 3 4 5 6 (% AUC) Initial NR 0.32 0.08 0.47 0.14 0.20 1.940° C./3 days NR 0.99 1.56 0.43 0.88 0 7.1 40° C./7 days NR 1.82 3.070.59 1.62 3.81 14.3 NR = not recorded

Example 24

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 42 was prepared.

TABLE 42 Ingredients Quantity Bivalirudin 5 mg/mL Sodium acetate 50 mMTween 80 0.02%   Sucrose 10% Propylene Glycol 10%

The samples were stored at 40° C. for 3 and 7 days and the stabilitydata is presented below in Table 43.

TABLE 43 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.radants Time 1 2 3 4 5 6 (% AUC) Initial NR 0.32 0.08 0.45 0.13 0.08 1.540° C./3 days NR 0.92 1.76 0.44 0.86 1.76 7.1 40° C./7 days NR 1.73 3.440.58 1.60 3.52 13.9 NR = not recorded

Example 25

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 44 was prepared.

TABLE 44 Ingredients Quantity Bivalirudin 5 mg/mL Sodium acetate 50 mMGlycine 0.5%  Tween 80 0.02%   Propylene glycol 10% Sodium hydroxide 1 Mq.s. to pH 4.5 Water q.s.

The samples were stored at 40° C. for 3 and 7 days and the stabilitydata is presented below in Table 45.

TABLE 45 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial NR 0.35 0.10 0.45 0.130.10 1.6 40° C./3 days NR 0.98 1.42 0.46 1.00 0.00 7.0 40° C./7 days NR1.79 2.79 0.65 1.74 3.45 13.0 NR = not recorded

Example 26

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 46 was prepared.

TABLE 46 Ingredients Quantity Bivalirudin 5 mg/mL Sodium acetate 50 mMGlycine 0.5%  Glycerol 10% Propylene glycol 10% Sodium hydroxide 1 Mq.s. to pH 4.5 Water q.s.

The samples were stored at 40° C. for 3 and 7 days and the stabilitydata is presented below in Table 47.

TABLE 47 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial NR 0.34 0.08 0.48 0.130.08 1.6 40° C./3 days NR 1.00 1.68 0.42 0.82 0.00 7.5 40° C./7 days NR1.95 3.32 0.49 1.44 3.88 14.3 NR = not recorded

Example 27

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 48 was prepared.

TABLE 48 Ingredients Quantity Bivalirudin 5 mg/mL Sodium acetate 50 mMTween 80 0.02%   Glycerol 10% Magnesium chloride 50 mM Sodium hydroxide1 M q.s. to pH 4.5 Water q.s.

The samples were stored at 40° C. for 3 and 7 days and the stabilitydata is presented below in Table 49.

TABLE 49 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial NR 0.32 0.08 0.47 0.140.09 1.6 40° C./3 days NR 0.99 1.40 0.51 1.07 0.00 7.3 40° C./7 days NR1.61 2.74 0.80 1.90 3.09 13.0 NR = not recorded

Example 28

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 50 was prepared.

TABLE 50 Ingredients Quantity Bivalirudin 5 mg/mL Sodium acetate 50 mMSucrose 10% Glycerol 10% Magnesium chloride 50 mM Propylene glycol 10%Sodium hydroxide 1 M q.s. to pH 4.5 Water q.s.

The samples were stored at 40° C. for 3 and 7 days and the stabilitydata is presented below in Table 51.

TABLE 51 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial NR 0.32 0.09 0.47 0.130.07 1.6 40° C./3 days NR 0.88 1.25 0.43 0.83 0.00 6.5 40° C./7 days NR1.59 2.47 0.53 1.61 3.48 12.7 NR = not recorded

Example 29

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 52 was prepared.

TABLE 52 Ingredients Quantity Bivalirudin 5.0 mg/mL PEG 400 10% Sodiumhydroxide 1 M q.s. to pH 4.5 Water q.s.

The samples were stored at 40° C. for 3 days and the stability data ispresented below in Table 53.

TABLE 53 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0 0 0 0.55 0.17 0.05 1.740° C./3 days 1.46 0 0.53 0.46 0.81 2.04 7.4

Example 30

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 54 was prepared.

TABLE 54 Ingredients Quantity Bivalirudin 5 mg/mL PEG 400 10% Sorbitol10% Calcium chloride 25 mM Sodium hydroxide 1 M q.s. to pH 4.5 Waterq.s.

The samples were stored at 40° C. for 3 days and the stability data ispresented below in Table 55.

TABLE 55 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0.04 0 0 0.53 0.17 0.052.0 40° C./3 days 1.05 0 0.36 0.48 0.90 1.69 6.5

Example 31

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 56 was prepared.

TABLE 56 Ingredients Quantity Bivalirudin 5.0 mg/mL PEG 400 20% Citricacid 50 mM Sorbitol 20% Calcium chloride 50 mM Poloxamer 0.6%  Sodiumhydroxide 1 M q.s. to pH 4.5 Water q.s.

The samples were stored at 40° C. for 3 days and the stability data ispresented below in Table 57.

TABLE 57 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0.06 0 0 0.49 0.16 0.071.9 40° C./3 days 1.04 0 0.24 0.41 0.65 1.61 7.5

Example 32

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 58 was prepared.

TABLE 58 Ingredients Quantity Bivalirudin 5 mg/mL Sorbitol 15% Magnesiumchloride 50 mM Sodium hydroxide 1 M q.s. to pH 4 Water q.s.

The samples were stored at 25° C. for 1 month and at 5° C. for 6 monthsand the stability data is presented below in Table 59.

TABLE 59 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0.03 0.05 0 0.51 0 0 1.525° C./1 month 0.41 1.05 0.45 0.33 0.29 1.42 6.6 5° C./6 months 0.540.62 0.18 0.32 0.33 0.63 4.2

Example 33

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 60 was prepared.

TABLE 60 Ingredients Quantity Bivalirudin 5 mg/mL Sorbitol 15% Propyleneglycol 10% Sodium hydroxide 1 M q.s. to pH 4.5 Water q.s.

The samples were stored at 25° C. for 1 month and at 5° C. for 6 monthsand the stability data is presented below in Table 61.

TABLE 61 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0.01 0.09 0.04 0.15 0 NR1.6 25° C./1 month 0.24 0.85 0.78 0.63 0.38 1.72 6.3 5° C./6 months 0.600.65 0.28 0.35 0.30 0.66 3.6 NR = not recorded

Example 34

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 62 was prepared.

TABLE 62 Ingredients Quantity Bivalirudin 5 mg/mL Sorbitol 15% Propyleneglycol 10% Sodium hydroxide 1 M q.s. to pH 4 Water q.s.

The samples were stored at 25° C. for 1 month and at 5° C. for 6 monthsand the stability data is presented below in Table 63.

TABLE 63 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0.03 0.04 0 0.18 0.09 NR1.9 25° C./1 month 0.43 1.14 0.52 0.50 0.52 1.53 7.8 5° C./6 months 0.610.68 0.17 0.21 0.15 0.57 4.1 NR = not recorded

Example 35

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 64 was prepared.

TABLE 64 Ingredients Quantity Bivalirudin 5 mg/mL Propylene glycol 10%Sodium hydroxide 1 M q.s. to pH 4 Water q.s.

The samples were stored at 25° C. for 1 month and at 5° C. for 6 monthsand the stability data is presented below in Table 65.

TABLE 65 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0.03 0.06 0 0.04 0 NR 1.825° C./1 month 0.41 1.14 0.63 0.46 0 1.66 5.9 5° C./6 months 0.64 0.750.15 0.29 0.28 0.69 3.8 NR = not recorded

Example 36

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 66 was prepared.

TABLE 66 Ingredients Quantity Bivalirudin 5 mg/mL PEG 400 20% Propyleneglycol 10% Sodium hydroxide 1 M q.s. to pH 4.5 Water q.s.

The samples were stored at 25° C. for 1 month and at 5° C. for 6 monthsand the stability data is presented below in Table 67.

TABLE 67 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0.23 0 0 0.13 0 NR 1.5 25°C./1 month 0.16 0.70 0.18 0.58 0.11 1.47 5.3 5° C./6 months 0.52 0.600.24 0.36 0.24 0.74 3.9 NR = not recorded

Example 37

In accordance with the general procedure of Example 3, the RTUbivalirudin composition shown in Table 68 was prepared.

TABLE 68 Ingredients Quantity Bivalirudin 5 mg/mL Sorbitol 15% PEG 40020% Sodium hydroxide 1 M q.s. to pH 5 Water q.s.

The samples were stored at 25° C. for 1 month and 5° C. for 6 months andthe stability data is presented below in Table 69.

TABLE 69 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0.26 0.18 0 0.14 0 NR 1.825° C./1 month 0 0.78 0 0.63 0.19 1.66 5.1 5° C./6 months 0.56 0.68 0.290.36 0.36 0.76 4.0 NR = not recorded

Example 38 General Procedure for the Preparation of 1 L RTU BivalirudinCompositions

A 2 M acetic acid solution was prepared by diluting 23.03 mL of aceticacid with water in a 200 mL volumetric flask. A 1 N NaOH solution wasprepared by dissolving 8 g of NaOH with water in a 200 mL volumetricflask. A 0.05 M sodium acetate solution was prepared by dissolving 13.68g of sodium acetate trihydrate in 1600 mL of water and adjusting the pHto 4.25 by the addition of 2 M acetic acid solution (approximately 100mL).

Bivalirudin (5.85 g) was slowly added with stirring to one part of thesodium acetate solution while maintaining the pH between about 4.15 and4.35. In the event the drug substance stuck to the glass beaker, 1 NNaOH solution was added (approximately 15 mL) until the pH was raised toabout 4.35. After all the drug substance was added, the pH of the drugsolution was adjusted to 4.25 with 2 M acetic acid (approximately 1.7mL).

The solution was filtered through a 0.22 μm sterilized filter and purgedwith nitrogen for about 10 min. The filtered drug solution wastransferred into 50 mL piggyback vials and 5 mL glass vials (SO₂treated, USP Type I flint glass vials). After additional nitrogenpurging (approximately 30 seconds for each vial), the vials were closedwith stoppers and crimped.

Example 39

In accordance with the general procedure of Example 38, the RTUbivalirudin composition shown in Table 70 was prepared.

TABLE 70 Ingredients Quantity Bivalirudin 5 mg/mL Sodium acetate 0.05 MAcetic acid 2 M Sodium hydroxide 1 M q.s. to pH 4.25 Water q.s.

The samples were stored at 5° C. for 3, 6, 9 and 12 months and at 25° C.for 1 month and the stability data is presented below in Table 71.

TABLE 71 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0.09 0.26 0.15 0.27 0.120.18 1.2 5° C./2 months 0.21 0.47 0.42 0.30 0.35 0.84 2.6 5° C./6 months0.13 0.66 0.72 0.28 0.51 1.44 4.2 5° C./9 months 0.11 0.87 1.44 0.331.40 2.00 6.4 5° C./12 months 0.24 1.18 1.87 0.49 1.04 2.73 8.5 25° C./1month 0.44 1.19 2.32 0.51 1.08 2.60 9.0

Example 40

In accordance with the general procedure of Example 38, the RTUbivalirudin composition shown in Table 72 was prepared.

TABLE 72 Ingredients Quantity Bivalirudin 5 mg/mL Sodium acetate 0.05 MAcetic acid 2 M Sodium hydroxide 1 M q.s. to pH 4.25 Sodium chloride 9mg/mL Water q.s.

The samples were stored at 5° C. for 2, 6, 9 and 12 months and at 25° C.for 1 month and the stability data is presented below in Table 73.

TABLE 73 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0.09 0.28 0.09 0.32 0.110.11 1.2 5° C./2 months 0.11 0.40 0.24 0.28 0.26 0.64 1.9 5° C./6 months0.17 0.67 0.58 0.31 0.55 1.46 4.2 5° C./9 months 0.10 0.85 0.94 0.281.39 1.74 5.6 5° C./12 months 0.18 1.14 1.25 0.43 0.93 2.51 7.4 25° C./1month 0.63 1.33 1.91 0.51 1.16 2.74 8.6

Example 41

In accordance with the general procedure of Example 38, the RTUbivalirudin composition shown in Table 74 was prepared.

TABLE 74 Ingredients Quantity Bivalirudin 5 mg/mL Sodium acetate 0.05 MAcetic acid 2 M q.s. to pH 4.25 Methyl paraben 0.1% Sodium chloride 9mg/mL Water q.s.

The samples were stored at 5° C. for 2, 6, 9 and 12 months and at 25° C.for 1 month and the stability data is presented below in Table 75.

TABLE 75 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0.09 0.26 0.08 0.35 0.11 01.0 5° C./2 months 0 0.39 0.22 0.26 0.33 0.62 2.1 5° C./6 months 0.060.66 0.52 0.28 0.93 1.51 4.4 5° C./9 months 0 0 0 0.37 1.36 2.14 6.1 5°C./12 months 0.17 1.32 1.48 0.47 1.02 2.81 8.2 25° C./1 month 0.79 1.321.94 0.52 1.22 2.88 9.1

Example 42

In accordance with the general procedure of Example 38, the RTUbivalirudin composition shown in Table 76 was prepared.

TABLE 76 Ingredients Quantity Bivalirudin 5 mg/mL Sodium acetate 0.05 ML-Histidine 1 mg/mL L-Methionine 1 mg/mL Acetic acid 2 M q.s. to pH 4.25Sodium chloride 9 mg/mL Water q.s.

The samples were stored at 5° C. for 2, 6, 9 and 12 months and at 25° C.for 1 month and the stability data is presented below in Table 77.

TABLE 77 Known Impurities (% AUC) Total Deg- Imp. Imp. Imp. Imp. Imp.Imp. radants Time 1 2 3 4 5 6 (% AUC) Initial 0 0.20 0.07 0.30 0.10 00.7 5° C./2 months 0.10 0.38 0.19 0.26 0.29 0.59 2.0 5° C./6 months 0.100.67 0.60 0.26 1.02 1.47 4.5 5° C./9 months 0.13 0.94 1.07 0.26 1.382.05 5.8 5° C./12 months 0.52 1.26 1.44 0.46 0.97 2.70 8.0 25° C./1month 0.48 1.40 1.91 0.50 1.17 2.71 8.7

Example 43

Prediction of Shelf-Life for Selected Formulations at about 5° C. Basedon % Bivalirudin

The shelf life of RTU bivalirudin compositions at about 5° C. waspredicted using an Arrhenius plot to project the amount of time untilthe bivalirudin content reached 90%. In addition, for some compositionsshelf life was predicted based on the stability data at 5° C. for 6months.

TABLE 78 Bivalirudin Content Example # (>90%) 32 ~16 months 33 ~24months 34 ~17 months 35 ~19 months 36 ~17 months 37 ~17 months 39 ~18months 40 ~19 months

The data from Table 78 suggests that a shelf life of greater than 16months may be attainable when the RTU bivalirudin compositions arestored at about 5° C.

1-20. (canceled)
 21. A method of preparing an aqueous, injectablebivalirudin composition that has not been reconstituted from alyophilizate comprising: (i) preparing a buffering agent having a pKa ofabout 2.5 to about 6.5; (ii) mixing an amount of bivalirudin, or saltsthereof, with the buffering agent to form a solution; (iii) adding apH-adjusting agent to the solution to adjust its pH to about 4 to lessthan 5; (iv) transferring the solution into one or more containers,wherein the concentration of bivalirudin in the containers is about 1mg/mL to about 10 mg/mL.
 22. The method of claim 21, wherein thebivalirudin in the containers is at a concentration of about 5 mg/mL.23. The method of claim 21, wherein the bivalirudin in the containers isat a concentration of about 10 mg/mL.
 24. The method of claim 21,wherein the buffering agent comprises acetate, tartrate, ascorbate,lactobionate, gentisate, succinate, lactate, α-lipoic acid, or anycombination thereof.
 25. The method of claim 24, wherein the bufferingagent is acetate.
 26. The method of claim 21, wherein the bufferingagent has a pKa of about 3.5 to about
 5. 27. The method of claim 26,wherein the buffering agent has a pKa of about 4.2 to about 4.5.
 28. Themethod of claim 21 wherein the pH-adjusting agent is an acid or base.29. The method of claim 28, wherein the pH-adjusting agent comprisesacetic acid or sodium hydroxide.
 30. The method of claim 21, wherein thepH is adjusted to between about 4.2 and about 4.5.
 31. The method ofclaim 21, further comprising adding at least one preservative prior totransferring the solution in step (iv).
 32. The method of claim 31,wherein the preservative is methyl-paraben.
 33. The method of claim 21,further comprising adding at least one antioxidant prior to transferringthe solution in step (iv).
 34. The method of claim 33, wherein theantioxidant is selected from a group consisting of histidine,methionine, and a combination thereof.
 35. The method of claim 21,further comprising sterilizing the solution prior to transferring thesolution in step (iv).
 36. The method of claim 35, wherein the solutionis sterilized through aseptic filtration.
 37. A method of preparing anaqueous, injectable bivalirudin composition that has not beenreconstituted from a lyophilizate comprising: (v) dissolving astabilizing agent selected from a group consisting of polymers, andsugars or polyols, in water; (vi) mixing an amount of bivalirudin, orsalts thereof, with the stabilizing agent to form a solution; (vii)adding a pH-adjusting agent to the solution to adjust its pH to about 4to less than 5; (viii) transferring the solution into one or morecontainers, wherein the concentration of bivalirudin in the containersis about 1 mg/mL to about 10 mg/mL.
 38. The method of claim 37, whereinthe bivalirudin in the containers is at a concentration of about 5mg/mL.
 39. The method of claim 37, wherein the bivalirudin in thecontainers is at a concentration of about 10 mg/mL.
 40. The method ofclaim 37, wherein the polymers are selected from a group consisting ofpolyethylene glycol, poloxamer, polysorbates, hydroxyethyl starch,polyvinylpyrrolidone, and any combination thereof.
 41. The method ofclaim 37, wherein the sugars or polyols are selected from a groupconsisting of sucrose, dextrose, dextrin, propylene glycol, sorbitol,glycerol, and any combination thereof.
 42. The method of claim 37,wherein the pH-adjusting agent is an acid, base, or buffering agent. 43.The method of claim 42, wherein the pH-adjusting agent comprises aceticacid or sodium hydroxide.
 44. The method of claim 37, wherein the pH isadjusted to between about 4.2 and about 4.5.
 45. The method of claim 37,further comprising adding at least one preservative prior totransferring the solution in step (iv).
 46. The method of claim 45,wherein the preservative is methyl-paraben.
 47. The method of claim 37,further comprising adding at least one antioxidant prior to transferringthe solution in step (iv).
 48. The method of claim 47, wherein theantioxidant is selected from a group consisting of histidine,methionine, and a combination thereof.
 49. The method of claim 37,further comprising sterilizing the solution prior to transferring thesolution in step (iv).
 50. The method of claim 49, wherein the solutionis sterilized through aseptic filtration.
 51. Use of an aqueous,injectable composition that has not been reconstituted from alyophilizate as an anticoagulant in a patient in need thereof, whereinthe composition comprises: bivalirudin (SEQ ID NO: 1), or salts thereof,at a concentration of about 1 mg/mL to about 10 mg/mL; (ii) one or morepharmaceutically acceptable stabilizing agents, wherein at least one ofthe stabilizing agents is a buffering agent having a pKa of about 2.5 toabout 6.5; (iii) a pH of about 4 to less than 5; and (iv) totalimpurities in an amount less than about 15% area-under-the-curve (“AUC”)as determined by high performance liquid chromatography (“HPLC”) afterstorage at 25° C. for 1 month.
 52. The use of claim 51, wherein thepatient is undergoing percutaneous coronary intervention (PCI).
 53. Theuse of claim 52, wherein the PCI is percutaneous transluminal coronaryangioplasty.
 54. Use of an aqueous, injectable composition that has notbeen reconstituted from a lyophilizate for the prevention of venousthromboembolic disease, wherein the composition comprises: (i)bivalirudin (SEQ ID NO: 1), or salts thereof, at a concentration ofabout 1 mg/mL to about 10 mg/mL; (ii) one or more pharmaceuticallyacceptable stabilizing agents, wherein at least one of the stabilizingagents is a buffering agent having a pKa of about 2.5 to about 6.5;(iii) a pH of about 4 to less than 5; and (v) total impurities in anamount less than about 15% area-under-the-curve (“AUC”) as determined byhigh performance liquid chromatography (“HPLC”) after storage at 25° C.for 1 month.
 55. Use of an aqueous, injectable composition that has notbeen reconstituted from a lyophilizate for the treatment of venousthromboembolic disease, wherein the composition comprises: (i)bivalirudin (SEQ ID NO: 1), or salts thereof, at a concentration ofabout 1 mg/mL to about 10 mg/mL; (ii) one or more pharmaceuticallyacceptable stabilizing agents, wherein at least one of the stabilizingagents is a buffering agent having a pKa of about 2.5 to about 6.5;(iii) a pH of about 4 to less than 5; and (vi) total impurities in anamount less than about 15% area-under-the-curve (“AUC”) as determined byhigh performance liquid chromatography (“HPLC”) after storage at 25° C.for 1 month.
 56. Use of an aqueous, injectable composition that has notbeen reconstituted from a lyophilizate for the treatment in patientswith unstable angina undergoing percutaneous transluminal coronaryangioplasty, wherein the composition comprises: (i) bivalirudin (SEQ IDNO: 1), or salts thereof, at a concentration of about 1 mg/mL to about10 mg/mL; (ii) one or more pharmaceutically acceptable stabilizingagents, wherein at least one of the stabilizing agents is a bufferingagent having a pKa of about 2.5 to about 6.5; (iii) a pH of about 4 toless than 5; and (vii) total impurities in an amount less than about 15%area-under-the-curve (“AUC”) as determined by high performance liquidchromatography (“HPLC”) after storage at 25° C. for 1 month.
 57. Use ofan aqueous, injectable composition that has not been reconstituted froma lyophilizate with a glycoprotein IIb/IIIa inhibitor for treatingpatients undergoing percutaneous coronary intervention, wherein thecomposition comprises: (i) bivalirudin (SEQ ID NO: 1), or salts thereof,at a concentration of about 1 mg/mL to about 10 mg/mL; (ii) one or morepharmaceutically acceptable stabilizing agents, wherein at least one ofthe stabilizing agents is a buffering agent having a pKa of about 2.5 toabout 6.5; (iii) a pH of about 4 to less than 5; and (viii) totalimpurities in an amount less than about 15% area-under-the-curve (“AUC”)as determined by high performance liquid chromatography (“HPLC”) afterstorage at 25° C. for 1 month.
 58. Use of an aqueous, injectablecomposition that has not been reconstituted from a lyophilizate fortreating patients with, or at risk of, heparin-induced thrombocytopeniaor heparin-induced thrombosis-thrombocytopenia syndrome undergoingpercutaneous coronary intervention, wherein the composition comprises:(i) bivalirudin (SEQ ID NO: 1), or salts thereof, at a concentration ofabout 1 mg/mL to about 10 mg/mL; (ii) one or more pharmaceuticallyacceptable stabilizing agents, wherein at least one of the stabilizingagents is a buffering agent having a pKa of about 2.5 to about 6.5;(iii) a pH of about 4 to less than 5; and (ix) total impurities in anamount less than about 15% area-under-the-curve (“AUC”) as determined byhigh performance liquid chromatography (“HPLC”) after storage at 25° C.for 1 month.